|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
The regulated secretory pathways is activated by protein kinase C (PKC) family members. However, the specific PKC isozymes involved in the pathway and the downstream of PKC activation are not clear. We have shown previously that Protein kinase C epsilon (nPKCepsilon) plays a key role in the basal and thyrotropin-releasing hormone-(TRH) stimulated prolactin (PRL) secretion in rat pituitary GH_4C_1 cells (Akita, Y., et.al. (1994) J.Biol.Chem.269,4653-4660). Here we examined the molecular mechanism by which nPKCepsilon mediates TRH-induced signal transduction. TRH exposure of GH_4C_1 cells immediately stimulated phosphorylation of six proteins, a p80 (M_r - 80,000 and pI - 4.3), three p52 (p52a, b and c ; M_r - 52,000 and pI - 5.75,5.7, and 5.6, respectively), and two p19 (p19a and b ; M_r - 19,000 and pI - 5.6 and 5.5, respectively), correlating with translocation of nPKCepsilon from the cytosol to the membrane/cytoskeletal fractions. Phorbol ester also enhanced these phosphorylations, w
hereas bisindolylmaleimide, a specific inhibitor of PKC,clearly inhibited the phosphorylation of p80 and p52. p80 and p19 were identified as myristoylated alanin-rich C kinase substrate (MARCKS) and stathmin, respectively, as assessed by the two-dimensional gel-electrophoretic profiles and their stabilities to heat and acid treatments. TRH stimulates the rapid redistribution of MARCKS from the membrane/cytoskeletal fraction to the cytosol fraction. Peptide mapping and subcellular fractionation indicated taht p52a, b, and c are the phosphorylated forms of a cytoskeletal protein (CP) with increasing solubilities, while the nonphosphorylated form, p52n, is tightly bound to the membrane/cytoskeletal fraction. In nPKCepsilon-overexpressing stable clones, the phosphorylated levels of MARCKS and CP but not stathmin wer enhanced and sustained upon TRH stimulation. These results suggest that two cytoskeletal proteins, MARCKS and CP,wer the major substrates of nPKCepsilon in vivo, and that these phosphrylations may regulate TRH-stimulated PRL secretion.
Moreover, we have studied on the role of protein kinase C (PKC) isozymes in degranulation of rat mast RBL-2H3 cells, and shown that cPKCbetaII acts stimulatory whereas nPKCepsilon acts inhibitory on the exocytosis of histamine-containing granule.
更に、PKCの活性化以降の作用機構を明らかにするために、PKCεを高発現したGH細胞株を樹立し、二次元電気泳動法によりPKC εの特異的基質を探索した。その結果、80kDと52kDの細胞骨格系の二種の蛋白質が、PRL分泌に関与している可能性を見出した。おそらく、これらの蛋白質のεによるリン酸化が細胞骨格の再編成を引き起こし、ホルモン分泌顆粒が細胞膜の開口放出の場に移行し易くなた結果、PRL分泌が上昇したと考えられる。 Less